PBX3 promotes pentose phosphate pathway and colorectal cancer progression by enhancing G6PD expression

Metabolic reprogramming is a hallmark of cancers crucial for fulfilling the needs of energy, building blocks, and antioxidants to support tumor cells' rapid proliferation and to cope with the harsh microenvironment. Pre-B-cell leukemia transcription factor 3 (PBX3) is a member of the PBX family whose expression is up-regulated in various tumors, however, whether it is involved in tumor cell metabolic reprogramming remains unclear. Herein, we report that PBX3 is a positive regulator of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway (PPP). PBX3 promoted G6PD transcriptional activity in tumor cells by binding directly to its promoter, leading to PPP stimulation and enhancing the production of nucleotides and NADPH, a crucial reductant, thereby promoting nucleic acid and lipid biosynthesis while decreasing intracellular reactive oxygen species levels. The PBX3/G6PD axis also promoted tumorigenic potential in vitro and in vivo. Collectively, these findings reveal a novel function of PBX3 as a regulator of G6PD, linking its oncogenic activity with tumor cell metabolic reprogramming, especially PPP. Furthermore, our results suggested that PBX3 is a potential target for metabolic-based anti-tumor therapeutic strategies.

Table S1.Primer pairs used for qRT-PCR.Table S2.Antibodies used for western blotting, ChIP assay, and immunohistochemistry.

RNA extraction and qRT-PCR
Total RNA was extracted using Trizol (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's instructions.Total RNA sample (1 g) was reversetranscribed into cDNA using the PrimeScript Reagent Kit with gDNA Eraser (Takara Bio, Dalian, China), then qRT-PCR was performed with SYBR Premix Ex Taq (Takara Bio) to assess mRNA expression levels.Primer sequences for qRT-PCR are shown in Supplementary Table S1.-actin was used to normalize sample amplification.The results are shown as relative to the expression level in the corresponding controls, which are assumed as 1.

Western blotting
Cells were collected and lysed with RIPA lysis buffer supplemented with a protease inhibitor and phosphatase inhibitor cocktail (complete cocktail; Roche Applied Science, Mannheim, Germany).An equal amount of samples (20 g) were subjected to electrophoresis on sodium dodecyl sulfide polyacrylamide gels and transferred to polyvinylidene (PVDF) fluoride membranes with a pore size of 0.45 m (Millipore, Billerica, MA).The membrane was then incubated with primary antibodies followed by secondary antibodies.Antibodies used are listed in Supplementary Table S2, and immunoblotting with anti--actin antibody was conducted to ensure equal protein loading.The signals were detected using the SuperSignal West Femto Maximum Sensitivity Substrate detection system (Thermo Scientific, Waltham, MA).Quantification was performed using Quantity One, and the result was normalized using -actin.

Apoptotic rate analysis
Cells were prepared as described above.Twenty-four hours after being re-seeded in 6well plates, cells were collected and stained with Annexin V/PI using Apoptosis Assay Kit (Biosharp, Hefei, China).The apoptotic rate was determined using flow cytometry.

Nile red staining
Cells were fixed using 4% paraformaldehyde and stained with 0.01 mg/ml Nile red (Sigma-Aldrich, St. Louis, MO) for 20 min.Nuclei were stained with DAPI.Images were taken using Olympus IX7I (Olympus, Tokyo, Japan).Quantitative analysis was performed using ImageJ (NIH, Bethesda, MD).Fluorescence intensities were normalized by the corresponding cell number.

5-ethynyl-2′-deoxyuridine (EdU) incorporation assay
Cells were prepared as described above.Twenty-four hours after being re-seeded in 24-well plates, cells were subjected to EdU incorporation assay using BeyoClick TM EdU Cell Proliferation Kit with Alexa Flour 488 (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instruction.Hoechst was used to stain the nuclei.Images were obtained using Olympus IX73 (Olympus).The ratio of de novo DNA synthesis was calculated as the ratio of EdU-positive cells to Hoechst-positive cells.
For the experiment with nucleosides, cells were re-seeded in 96-well plates at a density of 5 × 10 3 cells/well and cultured with medium containing 200 M (final concentration) mixture of 4 ribonucleosides (adenosine, uridine, cytidine, and guanosine) and 4 deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine) in the presence or absence of ROS scavenger Nacetyl-L-cysteine (final concentration: 2 M).Cell numbers were counted as described above three days after the cells were re-seeded.

Colony formation assay
Cells were prepared as described above, re-seeded into 6-well plates at a density of 300 cells/well, and cultured for 14 days.Cells were then fixed with 4% paraformaldehyde and stained with methylene blue.Quantification was then performed by counting the number of colonies formed.

Immunohistochemistry and hematoxylin-eosin (H&E) staining
Fresh human CRC tissue, normal adjacent tissue, and xenograft tumors were fixed overnight with 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 m using cryostat.Sections were then dewaxed using xylene, rehydrated, and subjected to immunohistochemical staining.Briefly, the tissue sections were incubated with primary antibodies for 1 h, followed by incubation with corresponding secondary antibodies conjugated with horse-radish peroxidase.
Visualization was performed using a DAB Kit (DAKO, Beijing, China) under a microscope.The nuclei were then counterstained with hematoxylin (Beyotime Biotechnology), followed by dehydration and coverslip mounting.The antibodies used were listed in Supplementary Table S2.Images were taken using Pannoramic Midi (3DHistech, Budapest, Hungary).
For hematoxylin-eosin (H&E) staining, paraffin sections from human colon cancer tissues, normal adjacent tissues, and mice subcutaneous tumors generated in xenograft experiment (4 m thickness) were fixed in 10% formalin and washed with 60% propylene glycerol.The samples were stained with 0.5% hematoxylin-eosin (Sangon Bio, Shanghai, China) for 3 min followed by dehydration and coverslip mounting.Images were taken using Pannoramic Midi.

Dual luciferase reporter assay
Cells were co-transfected with indicated vectors, reporter vector bringing the firefly luciferase, and Renilla luciferase expression vector pRL-SV40 (Promega).After 24 h, luciferase activities were analyzed using the Dual Luciferase Reporter Assay (Promega).The activities of the firefly luciferase reporters were normalized using those of Renilla luciferase.

Chromatin immunoprecipitation (ChIP) assay
Chromatin was immunoprecipitated using a ChIP Assay Kit (Beyotime Biotechnology) according to the manufacturer's instructions.In brief, after the cells were lysed, chromatins were then immunoprecipitated using protein A+G Agarose/salmon sperm DNA and anti-PBX3 antibody, anti-Histone H3 antibody, or normal rabbit IgG, and de-crosslinked for 4 h at 65 °C.After being treated with 0.5 M EDTA, 1 M Tris (pH 6.5), and 20 mg/ml proteinase K, immunoprecipitated chromatin was then subjected to PCR by using PrimeSTAR Max (Takara Bio).The sequence of the forward primer used was 5'-TGT GAA TAT ACA TAC AAC AAA CCA T-3'; while that of the reverse primer was 5'-GCA CCA TCA CTC CCA GCT A-3'.

Figure S3 .
Figure S3.Effect of PBX3 on intracellular NADPH and NADP + levels.

Figure S4 .
Figure S4.Effect of PBX3 overexpression on tumor cells lipid accumulation.

Figure S5 .
Figure S5.G6PD is crucial for PBX3 regulation on NADPH and NADP + levels.